The primary goal of the Phase I SBIR projects is to determine the feasibility of a novel neuraminidase assay to be used as an influenza neuraminidase inhibition and drug susceptibility assay. The proposed assay is a biochemiluminescence assay that uses a novel substrate and permits real time determination of neuraminidase activity, which essentially requires only one preparation step - the addition of sample to a master detection mix that contains all necessary reagents for the assay. The proposed efforts include 1) improving substrate synthesis, 2) evaluating the feasibility as a NI and drug susceptibility assay, and 3) determining whether the assay has sufficient sensitivity so that clinical specimens can be directly used for the assay. Two neuraminidase inhibitors - zanamivir and particularly oseltamivir - are the current mainstay of pharmacological intervention during an influenza epidemic and, if happened, a pandemic. Emergence of drug resistant influenza virus variants heightens the concerns about widespread use of these drugs. It is imperative to monitor the emergence of antiviral drug resistant variants. Both cellular and biochemical based assays have been used to determine viral susceptibility to neuraminidase inhibitors. Cellular assays suffer from a number of drawbacks, including selection of mutations that compensate for neuraminidase inhibitor resistance during cell culture, long assay time, and tedious procedures, which make them less suitable for large scale surveillance programs. In contrast, the biochemical assays directly measure the inhibition of viral neuraminidase activities, thus circumventing many of the problems associated with the cellular assays. However, current biochemical assays are still complicated, take hours to complete, and have significant assay variability. More importantly, they lack sufficient sensitivity to allow drug susceptibility determination directly using clinical specimens (e.g., nasal wash), a much preferred sample type for large scale surveillance programs. Successful development of the proposed assay will allow the use of clinical specimens for drug susceptibility testing - a significant improvement over the currently used assays, which have to use virus isolates that are obtained via the lengthy and cumbersome culture methods. PUBLIC HEALTH RELEVANCE: Two neuraminidase inhibitors - zanamivir and oseltamivir - are the current mainstay of pharmacological intervention during an influenza epidemic or pandemic. Given the history of emergence of drug resistant influenza virus variants, it is imperative to monitor the emergence of antiviral drug resistant variants, which requires a robust neuraminidase inhibition (NI) assay. The proposed efforts are to determine the feasibility of a novel neuraminidase activity assay as an influenza NI assay. Specifically, we will determine if the assay is 1) rapid (5 min preparation time plus 30 min incubation);2) simple (essentially one step operation);3) sensitive (equivalent to or better than 25 cycles of real time PCR assay) so that clinical specimens can be directly used for NI assay;4) reproducible (CV<15%);and 5) suitable for a 96-well format for large-scale sample detection.